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Updated: Oct 19, 2025
Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting
Published on: May 2, 2025
Combinatorial CRISPR/Cas9 Screening Reveals Epistatic Networks of Interacting Tumor Suppressor Genes and Therapeutic
Xiaoyu Zhao1,2, Jinyu Li1, Zhimin Liu2,3,4,5
1Department of Pathology and Cancer Center, Renaissance School of Medicine, Stony Brook, New York.
Abstract:
The majority of cancers are driven by multiple genetic alterations, but how these changes collaborate during tumorigenesis remains largely unknown. To gain mechanistic insights into tumor-promoting genetic interactions among tumor suppressor genes (TSG), we conducted combinatorial CRISPR screening coupled with single-cell transcriptomic profiling in human mammary epithelial cells. As expected, different driver gene alterations in mammary epithelial cells influenced the repertoire of tumor suppressor alterations capable of inducing tumor formation. More surprisingly, TSG interaction networks were comprised of numerous cliques-sets of three or four genes such that each TSG within the clique showed oncogenic cooperation with all other genes in the clique. Genetic interaction profiling indicated that the predominant cooperating TSGs shared overlapping functions rather than distinct or complementary functions. Single-cell transcriptomic profiling of CRISPR double knockouts revealed that cooperating TSGs that synergized in promoting tumorigenesis and growth factor independence showed transcriptional epistasis, whereas noncooperating TSGs did not. These epistatic transcriptional changes, both buffering and synergistic, affected expression of oncogenic mediators and therapeutic targets, including CDK4, SRPK1, and DNMT1. Importantly, the epistatic expression alterations caused by dual inactivation of TSGs in this system, such as PTEN and TP53, were also observed in patient tumors, establishing the relevance of these findings to human breast cancer. An estimated 50% of differentially expressed genes in breast cancer are controlled by epistatic interactions. Overall, our study indicates that transcriptional epistasis is a central aspect of multigenic breast cancer progression and outlines methodologies to uncover driver gene epistatic networks in other human cancers. SIGNIFICANCE: This study provides a roadmap for moving beyond discovery and development of therapeutic strategies based on single driver gene analysis to discovery based on interactions between multiple driver genes.See related commentary by Fong et al., p. 6078.
Insights
Understanding how multiple genetic changes drive cancer is key. This study reveals that cooperating tumor suppressor genes (TSGs) in breast cancer often share functions and exhibit transcriptional epistasis, impacting therapeutic targets and patient tumors.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Most cancers involve multiple genetic alterations, but their collaborative roles in tumor development are poorly understood.
- Investigating genetic interactions among tumor suppressor genes (TSGs) is crucial for deciphering complex tumorigenesis.
Purpose of the Study:
- To elucidate the mechanistic insights into tumor-promoting genetic interactions among TSGs.
- To identify how combinatorial TSG alterations collaborate during cancer development.
Main Methods:
- Utilized combinatorial CRISPR screening in human mammary epithelial cells.
- Integrated single-cell transcriptomic profiling to analyze gene interactions and transcriptional changes.
- Performed genetic interaction profiling to map TSG networks.
Main Results:
- Identified TSG interaction networks forming "cliques" where genes within a set cooperate oncogenically.
- Demonstrated that cooperating TSGs predominantly share overlapping functions.
- Observed transcriptional epistasis in cooperating TSGs that synergize tumorigenesis, affecting key mediators like CDK4, SRPK1, and DNMT1.
- Validated epistatic alterations in human breast cancer patient tumors, with an estimated 50% of differentially expressed genes influenced by these interactions.
Conclusions:
- Transcriptional epistasis is a central mechanism in multigenic breast cancer progression.
- Cooperating TSGs exhibit functional overlap and drive cancer through synergistic transcriptional effects.
- This study provides a framework for developing therapeutic strategies targeting multiple driver gene interactions in cancer.
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